In vivo assessment of artery smooth muscle [Ca²⁺]_i and MLCK activation in FRET-based biosensor mice

The cellular mechanisms that control arterial diameter in vivo, particularly in hypertension, are uncertain. Here, we report a method that permits arterial intracellular Ca²⁺ concentration ([Ca ²⁺]_i), myosin light-chain kinase (MLCK) activation, and artery external diameter to be recorded simultaneously with arterial blood pressure (BP) in living mice under 1.5% isofluorane anesthesia. The method also enables an assessment of local receptor activity on [Ca²⁺] _i, MLCK activity, and diameter in arteries, uncomplicated by systemic effects. Transgenic mice that express, in smooth muscle, a Ca ²⁺/calmodulin-activated, Förster resonance energy transfer (FRET)-based "ratiometric", exogenous MLCK biosensor were used. Vasoactive substances were administered either intravenously or locally to segments of exposed femoral or cremaster arteries. In the basal state, mean BP was ∼90 mmHg, femoral arteries were constricted to 65% of their passive diameter, MLCK fractional activation was 0.14, and [Ca²⁺]i was 131 nM. Phenylephrine (300 ng/g wt iv) elevated mean BP transiently to ∼110 mmHg, decreased heart rate, increased femoral artery [Ca²⁺] _i to 244 nM and fractional MLCK activation to 0.24, and decreased artery diameter by 23%. In comparison, local application of 1.0 μM phenylephrine raised [Ca²⁺]_i to 279 nM and fractional MLCK activation to 0.26, and reduced diameter by 25%, but did not affect BP or heart rate. Intravital FRET imaging of exogenous MLCK biosensor mice permits quantification of changes in [Ca²⁺]_i and MLCK activation that accompany small changes in BP. Based on the observed variance of the FRET data, this method should enable the detection of a difference in basal [Ca ²⁺]_i of 29 nM between two groups of 12 mice with a significance of P < 0.05.